Background <p>Grape production generates substantial agricultural waste, particularly grape pomace, a by-product rich in bioactive compounds. Moreover, its reuse is strongly recommended to prevent environmental issues from improper disposal. This study explores the feasibility of using exhausted grape pomace, alone and in combination with anaerobic digestate, as substrates for <i>Pleurotus</i> mushroom cultivation. Grape pomace is rich in fiber, lipids, sugars, proteins, and phenolic compounds, which makes it a promising substrate for this purpose.</p> Results <p>Elemental analyses of this substrate revealed high carbon (46%) and nitrogen (2.5%) contents, along with greater levels of macro and microelements (P, Ca, Mg, Fe) compared to conventional substrates. Fourier Transformed Infrared (FTIR) spectroscopy identified aliphatic groups from seed fatty acids and polysaccharide bands, typical of the seedless part of grape pomace. Fatty acid evaluation confirmed the presence of partial glycerides with linoleic acid content exceeding 62%, while phenolic composition analysis highlighted a predominance of bound phenols. These chemical characteristics significantly increased mushroom production, with statistically significant increases in fresh weights observed upon the addition of grape pomace, resulting in variations exceeding 130% in some instances. The mixture with digestate exhibited the most promising results, probably due to improved pH values. FTIR and Surface Enhanced Raman Scattering (SERS) spectra of mushrooms grown on different matrices were similar, but with some differences likely related to substrate composition. In addition, SERS analysis identified ergothioneine, a significant fungal amino acid, which was not detectable in the FTIR spectra.</p> Conclusions <p>This study highlights grape pomace as an effective and sustainable substrate for mushroom cultivation, offering a viable alternative to conventional growth mediums. The high yield and consistent quality of mushrooms grown on grape pomace, particularly when mixed with anaerobic digestate, demonstrate its potential for commercial mushroom production. The findings support the broader goals of sustainable agriculture by promoting the recycling of agricultural by-products, minimizing waste, and adding economic value. This innovative approach to utilizing grape pomace contributes to improved environmental management and opens new opportunities for enhancing the efficiency and sustainability of mushroom cultivation practices.</p> Graphical Abstract <p></p>